Dataset Information

CRISPR/Cas9-RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae.

ABSTRACT: The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.

SUBMITTER: Kildegaard KR

PROVIDER: S-EPMC6619288 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae.

Kildegaard Kanchana Rueksomtawin KR Tramontin Larissa Ribeiro Ramos LRR Chekina Ksenia K Li Mingji M Goedecke Tobias Justus TJ Kristensen Mette M Borodina Irina I

Yeast (Chichester, England) 20190501 5

The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic target ...[more]

PMID: 30953378

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Project description:BackgroundThe abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers.ResultsThe aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L- 1 after 48 h under oxygen limited condition in bioreactor fermentations.ConclusionThis work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Dataset Information

CRISPR/Cas9-RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae.

Publications

CRISPR/Cas9-RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae.

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